Cancer of the cervix is the most common cancer in developing countries and the second most common in women worldwide (Parkin et al., 1993). Papillomaviruses are associated with greater than 90% of all cases of cervical cancers. Over 70 human papillomavirus (HPV) types have been identified with subsets of viruses that are predominately associated with malignant or benign neoplasias of the anogenital area, particularly the cervix (Salzman et al., 1987).
Although human papillomaviruses (HPVs) are implicated in the etiology of anogenital cancers, the complete story is not yet known. What is suspected is that expression of the HPV E6 and E7 oncoproteins may contribute to the carcinogenic process. Progressive loss of the ability to differentiate, and resistance to the growth-inhibitory effects of endogenous signals, also appear to be important in multistep tumorigenesis.
To develop therapeutic and diagnostic strategies directed toward papillomavirus caused diseases, the virus must be available for analysis. The study of the human pathogenic papillomaviruses (HPVs) and the ability to manipulate the virus for use in diagnostic assays or vaccines, has been hampered by the inability to propagate the virus in tissue culture. However, some progress has been made.
Because the replication cycle of papillomaviruses is intimately associated with the proliferation/differentiation cycles of terminally differentiating epithelium, it has been particularly difficult to cultivate and analyze various types of HPVs in vitro. Following infection of proliferating basal cells in epithelial tissues, papillomavirus replicates its genome to approximately 50 to 200 copies and maintains this copy number in basal cells during the productive life cycle of the virus. When a basal cell divides, one of the daughter cells migrates upwards and begins the process of terminal differentiation. The signal transduction and gene expression pathways that are elicited during the differentiation process regulate viral gene expression. Concomitant with the differentiation process, papillomavirus genes are expressed, viral DNA is amplified, viral late proteins are translated, and virion morphogenesis occurs. Papillomavirions are sloughed off with the squames of the outermost epidermal layer and can then infect another host. Because of this dependency on terminal epithelial differentiation for replication, papillomaviruses cannot grow in ordinary monolayer culture; this limitation has hindered studies on the papillomavirus life cycle.
The organotypic (collogen raft) tissue culture system accurately mimics an in vivo cellular environment and architecture (Ozbun and Meyers, 1996). Uses of the system are exemplified by investigations of the TGF-.beta.1 response of HPV-positive keratinocytes derived from neoplastic cervical biopsies. For example, transforming growth factor .beta.1 (TGF-.beta.1) is a potent growth inhibitor for a variety of cultured cells. However, there have been conflicting reports on the ability of TGF-.beta.1 to inhibit the growth of HPV-positive keratinocytes in monolayer cultures. Using organotypic raft cultures, some of these conflicts were elucidated (Ozburn et al., 1996).
The addition of 12-O-tetradecanoyl phorbol-13-acetate to the media of organotypic (raft) cultures increased expression of physiological markers of keratinocyte differentiation and concomitantly induced production of virions. Capsid production was detected in differentiated suprabasal cells. Virions approximately 54 nanometers in size were observed by electron microscopy in cross sections of raft tissue in the suprabasal layers. Virions purified through isopycnic gradients were found to contain type 31b DNA and exhibited an icosahedral shape similar to that of papillomaviruses found in clinical samples (Meyers et al., 1992).
The differentiation-specific amplification of episomal viral DNA, late gene expression, and virion morphogenic stages of HPV31 replication have been achieved in organotypic or (collagen raft,) cultures (Meyers et al., 1992; Frattini et al., 1996). However, culture systems continue to be deficient in the ability to propagate stocks of HPV competent for replication and infectivity studies. Additionally, investigation of the complete differentiation-dependent life cycle of HPV in vitro has been limited to a single viral type, HPV31. Another problem is the dependency on neoplastic biopsy material to derive cell lines. High risk HPV-contained cell lines established from biopsy material and primary cells transfected with a complete viral genome generally harbor HPV genomes that are integrated into the host DNA therefore are impaired in their ability to carry out a complete viral life cycle.
New methods are needed for in vitro viral propagation systems in particular for production of infectious viruses.